Spin-trapping of the superoxide free radical has provided ESR evidence of the formation of this species in microsomal incubations of adriamycin, daunorubicin, and mitomycin C. The time sequence of the appearance of the spin-trapped superoxide and the semiquinone radical metabolite of these quinone-containing anticancer drugs indicates that air oxidation of the semiquinone metabolite is responsible for the superoxide formation. Superoxide dismutase prevents the formation of the superoxide spin adducts. Microsomal incubations containing DNA-intercalated anthracyclines produce much less superoxide than incubations without DNA. The first clear ESR evidence for the semiquinone metabolite of mitomycin C in a biological system is presented.